Repeated high intensity laser biopsy pulses do not alter genetic testing results nor increases mosaicism following human embryo blastocyst biopsy of trophectoderm cells

Objective Recent data suggests that the rate of mosaic diagnosed embryos is increasing following Preimplantation Genetic Testing for Aneuploidy (PGT-A) when using Next Generation Sequencing (NGS). Trophectoderm cell biopsy by laser ablation is proposed as a possible cause of mosaic diagnoses resulting in the subsequent removal of possible genetically normal embryos from availability for embryo transfer. This study investigates whether repeated high intensity laser pulses during trophectoderm laser ablation alters the genetic results or causes increased mosaicism. Design Several trophectoderm biopsied cell samples from each embryo were exposed to either repeated laser pulses or increasingly intense pulses prior to NGS analysis for genetic results. Material and Methods Embryos were obtained from patients that consented to discarding their surplus embryos. Two genetically undiagnosed blastocysts were used for this study. Each embryo was subjected to low and high intensity laser pulses for varied pulse durations using the Hamilton Thorne Lykos laser. From both embryos 3-5 biopsied cell samples were obtained for each of the laser test categories. Samples were then exposed to direct and repeated laser pulses for 10 or 20 pulses @ 290µs; or 20 or 40 laser pulses @ 400µs. One additional sample, served as the control, and was exposed only to the standard biopsy procedure of 5 pulses @ 290µs. All samples were then washed through several washing buffer droplets prior to placement in PCR tubes in 2-3µl buffer and frozen at -20°C until NGS analysis was performed. Results This study demonstrated that genetic analysis results obtained from the control and increased intensity samples show no difference, with all providing normal genetic results (46, XY) (Table 1). Importantly, no mosaic genetic results were observed even at the maximum number of pulses or intensity level. Conclusion This study provides significant evidence that multiple laser pulses, even with increased laser intensities, do not alter the genetic results obtained when performing PGT-A. Additionally, the laser intensity does not cause incidences of mosaicism in the NGS results. To minimize potential damage to the embryo and the biopsied cells, optimal excision of the trophectoderm should be achieved using a limited number of laser pulses at the lowest intensity possible. It is important to note, that extreme repeated laser pulses, with increased intensity, may have a detrimental impact on the actual embryo which could reduce implantation potential of the embryo itself.